William H. Oldendorf

N-acetyl-L-aspartic acid: a literature review of a compound prominent in 1H-NMR spectroscopic studies of brain.

N-acetyl aspartic acid (NAA), discovered in 1956 by Tallan, is the major peak seen in water-suppressed NMR proton (hydrogen) spectroscopy. NAA makes up about one thousandth of the wet weight of human brain and appears to be limited solely to neurons. This compound has been shown to be relatively stable for a period of twenty-four hours post-mortem and the concentration of NAA is not changed by insulin-induced hypoglycemia. MAO inhibitors lower its concentration while reserpine and other drugs increase it. NAA has been implicated in many processes of the nervous system: it may be involved in the regulation of neuronal protein synthesis, myelin production, or the metabolism of several neurotransmitters such as aspartate or N-acetyl-aspartyl-glutamate. It is involved in the neurologic disorder Canavan disease and has grown to be a vital component of in vivo 1H-NMR spectroscopic studies.

TRANSPORT OF METABOLIC SUBSTRATES THROUGH THE BLOOD-BRAIN BARRIER'

THE ENDOTHELIAL cells of cerebral capillaries, like epithelial cells, possess tight junctions between the plasmalemma of adjacent cells (BRIGHTMAN et al., 1970). As a consequence, the plasma membranes of brain endothelial cells form a continuous membranous barrier between blood and brain interstitium. Similar to other cell membrane systems, the flux of circulating substances through the blood-brain barrier (BBB) occurs, via either (i) lipid mediation, or (ii) carrier mediation (OLDENDORF, 1976). During the past decade with the introduction of newer techniques such as tissue sampling, single-injection methodology (OLDENDORF, 1970), much quantitative information has been obtained regarding the carrier-mediated transport of metabolic substrates through the BBB. Plasma concentrations, transport K,, and V,,, estimates for substrates transported by one of four independent carrier systems (hexose, neutral amino acid, basic amino acid and monocarboxylic acid) have been recently tabulated (PARDRIDGE et ul., 1975; OLDENWRF, 1976). In addition, four other independent carrier systems have recently been identified; these systems mediate the transport into brain of nucleosides, purines (CORNFORD & OLDENDORF, 1975), acidic amino acids (OLDENDORF & SZABO, 1976), and choline (OLDENDORF & BRAUN, 1976). The 8 independent transport systems are listed in Table 1 relative to the V,,, estimate for each system. If it is assumed that each carrier moves through the membrane at a comparable rate, then the Vma,,,provides a measure of the redundancy of the transport system within the BBB. Generally, as barrier transport capacity (V,,,) decreases, the transport affinity increases, as represented by decreasing K , (Table 1). The non-saturable component of BBB transport of metabolic substrates varies over a 30-fold range (Table 1) and probably reflects transport via either very low affinity, high capacity systems, or via free diffusion.

Lipid Solubility and Drug Penetration of the Blood Brain Barrier

Summary Lipid/water partition coefficients of 19 radiolabeled drugs were correlated with uptake by brain during a single microcirculatory passage following carotid arterial injection. Uptake was measured relative to a simultaneously injected diffusible reference. When the lipid/ water partition coefficient was greater than about 0.03 a substantial fraction of the drug penetrated the blood-brain barrier and for most drugs above this range, uptake was essentially complete. These data suggest there is probably little reason to greatly exceed this degree of lipid solubility when designing drugs for blood-brain barrier penetration and central nervous system effects. Valuable discussions with Dr. Arthur K. Cho and Dr. Jared M. Diamond are acknowledged. Helpful suggestions from Mrs. Stella Z. Oldendorf were received, and technical assistance was provided by Mrs. Shigeyo Hyman and Mr. Leon Braun. Support is also acknowledged from the NIH-NINDS Project Grant No. NS 8711 and the Veterans Administration.

Blood-Brain Barrier: Penetration of Morphine, Codeine, Heroin, and Methadone after Carotid Injection

Labeled morphine, codeine, heroin, or methadone was injected as a bolus into the common carotid artery of the rat, and the rat was decapitated 15 seconds later. The brain uptake of the drug was calculated by measurement of the brain content of the drug as a percentage of a labeled, highly diffusible reference substance simultaneously injected. The uptake of morphine was below measurability; the uptake of codeine was 24 percent; heroin, 68 percent; and methadone, 42 percent. Brain uptakes of morphine and codeine were also studied after intravenous injection and correlated well with uptakes after carotid injection; the uptake of codeine being nearly complete by 30 seconds. These studies indicate that brain uptake of certain of these drugs is very rapid and that uptake of heroin injected intravenously is probably limited by the regional flow of blood in the brain. The possible relation of this rapid penetration of the blood-brain barrier by heroin to its strongly addictive properties is discussed.

Kinetic analysis of blood-brain barrier transport of amino acids.

The Michaelis-Menten kinetics of blood-brain barrier transport of fourteen amino acids was investigated with a tissue-sampling, single-injection technique in the anesthetized rat. Tracer quantities of 14C-labelled amino acids and 3H2O, used as a freely diffusible internal reference, were mixed in 0.2 ml of buffered Ringers solution and injected rapidly into a common carotid artery. Circulation was terminated by decapitation at 15s following injection. A brain uptake index (Ib) was determined from the ratio of 14C dpm in the brain tissue and the injection mixture divided by the same ratio for the 3H2O reference. Brain clearance of tracer concentration of amino acid was saturable when various concentrations of unlabeled amino acid were added to the injection solution. Double reciprocal plots of the saturation data yielded Km (mM) values that ranged from a low of 0.09 mM for arginine to a high of 0.75 mM for cycloleucine. Transport V values were determined from the relationship P = V/Km where P is the blood-brain barrier permeability constant (ml/g per min): P was calculated from the Ib for each amino acid based on a cerebral blood flow of 0.56 ml/g per min and a fractional extraction of 0.75 for the 3H2O reference 15s following carotid injection. The V values ranged from a low of 6.2 nmol/g per min for lysine to a high of 64 nmol/g per min for l-DOPA. Efflux of the tracer amino acid during the 15-s period after injection was assumed to be slow, since the rate constant of cycloleucine from brain to blood was low, 0.11 min-1.

KINETICS OF BLOOD‐BRAIN BARRIER TRANSPORT OF PYRUVATE, LACTATE AND GLUCOSE IN SUCKLING, WEANLING AND ADULT RATS

Abstract— The kinetics of the uptake from blood to brain of pyruvate, lactate and glucose have been determined in rats of different ages. The carotid artery single injection technique was used in animals anaesthetized with pentobarbital. The rates of influx for each substrate were determined over a range of concentrations for the different age‐groups. Data were analysed in terms of the Michaelis‐Menten equation with a component to allow for non‐saturable diffusion. Values are given for Km, Vmax and Kd. In suckling rats (15‐21 days) the Vmax values for both pyruvate and lactate were 2.0 μmol g−1 min−1. In 28‐day‐old rats the Vmax values had fallen to one‐half and in adults they were less than one‐tenth. Km, values were higher in the younger animals. The rate of glucose transport in suckling rats was half that of 28‐day‐old and adults although there was no difference with age in the Km values.

Kinetics of blood-brain barrier transport of hexoses

1. The kinetics of transport of glucose and four other hexoses through the blood-brain barrier were studied with a tritiated-water reference technique in the anesthetized rat. Brain clearance of [14-C]hexose was measured 15 s after a single injection of the hexose and 3-HOH reference into the common carotid artery. 2. Saturation of brain clearance of [14-C]hexose conformed to Michaelis-Menten kinetics. Linear transformation of the uptake data yielded the Km of carrier-mediated hexose transport: 2-deoxy-D-glucose 6 mM, D-glucose 9mM, 3-O-methyl-D-glucose and D-galactose 40 mM. A maximum transport velocity of 1.56 mumol/g per min was calculated and shown to be constant for all five hexoses. 3. The kinetics of 3-HOH and 3-0-methyl-D-[14-C]glucose efflux from brain to blood were studied with a modification of the water reference technique. An estimate of cerebral blood flow, 0.56 ml/g per min, was made from the efflux rate constant for 3-HOH, 0.61 min-1. The fractional extraction of 3-0-methyl-D-[14C]glucose uptake from blood was estimated from the efflux rate constant, 0.22 min-1, for this sugar and found to be 0.25. This value approximated the fractional extraction of 3-0-methyl-D-[14-C]glucose uptake that was determined from influx studies (0.24). These results indicated that the bidirectional movement of glucose across the blood brain barrier was symmetrical, which suggested that barrier sugar transport is equilibrative and not active. 4. Blood-brain barrier sugar transport was shown to be reversibly inhibited by phloretin, yet no modulation of transport was demonstrable after 2 or 8 days of starvation. Finally, regional analysis (olfactory bulb, caudate-putamen nucleus, thalamus-hypothalamus, and inferior-superior colliculi) demonstrated that, in addition to blood-brain barrier permeability, brain clearance of glucose was a function of cerebral blood flow.

Independent blood-brain barrier transport systems for nucleic acid precursors

The blood-brain barrier permeability to certain 14-C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15s later. The amount of 14-C-labelled base or nucleoside remaining in brain was expressed in relation to 3-H2O (a highly diffusible internal standard) and 113m-In-labelled EDTA (an essentially non-diffusible internal standard). Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine. Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (Km equals 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (Km equals 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [14-D]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.

Rapid appearance of intraventricularly administered neuropeptides in the peripheral circulation

125I-Labeled cholecystokinin octapeptide ([125I]CCK-OP) diffuses rapidly by a non-carrier-mediated mechanism into the peripheral blood following injection into the lateral ventricle of a rabbit. In contrast, no [125I]CCK-OP was observed in the CSF following intravenous injection. This unidirectional free transport mechanism from CSF to blood may apply to other neuropeptides as well.

Carrier mediated blood-brain barrier transport of choline and certain choline analogs.

Blood‐brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a Km= 442 ± 60 μM and Vmax= 10.0 ± 0.6 nmol min‐1 g‐1. In 14–15‐day‐old suckling forebrains a similar Km (= 404 ± 88 μM) but higher Vmax (= 12.5 ± 1.5 nmol min‐1 g‐1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis‐Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex Km= 310 ± 103 μM and Vmax= 12.6 ± 2.8 nmol min‐1g‐1; paleocortex Km= 217 ± 76 μM and Vmax= 7.2 ± 1.5 nmol min‐1 g‐1).